Energetics of Sequence-Specific Protein-DNA Association: Binding of Integrase Tn916 to Its Target DNA
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- 作者：Stoyan Milev ; Alemayehu A. Gorfe ; Andrey Karshikoff ; Robert T. Clubb ; Hans Rudolf Bosshard ; and Ilian Jelesarov
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：April 1, 2003
- 年：2003
- 卷：42
- 期：12
- 页码：3481 - 3491
- 全文大小：180K
- 年卷期：v.42,no.12(April 1, 2003)
- ISSN：1520-4995
文摘
The DNA binding domain of the transposon Tn916 integrase (INT-DBD) binds to its DNAtarget site by positioning the face of a three-stranded antiparallel 
-sheet within the major groove. Bindingof INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry,differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, andfluorescence spectroscopy. The observed heat capacity change accompanying the association reaction(
Cp) is temperature-dependent, decreasing from -1.4 kJ K-1 mol-1 at 4 
C to -2.9 kJ K-1 mol-1 at 30C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and theprotein-DNA complex are not changing in parallel when the temperature increases and that thermalmotions of the protein and the DNA are restricted in the complex. After correction for this effect, Cp is-1.8 kJ K-1 mol-1 and temperature-independent. However, this value is still higher than Cp of -1.2 kJK-1 mol-1 estimated by semiempirical methods from dehydration of surface area buried at the complexinterface. We propose that the discrepancy between the measured and the structure-based prediction ofbinding energetics is caused by incomplete dehydration of polar groups in the complex. In support, weidentify cavities at the interface that are large enough to accommodate ~10 water molecules. Our resultshighlight the difficulties of structure-based prediction of Cp (and other thermodynamic parameters) andemphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complexbut also of the folding thermodynamics of the protein and the DNA alone, which are described in theaccompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].
-sheet within the major groove. Bindingof INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry,differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, andfluorescence spectroscopy. The observed heat capacity change accompanying the association reaction(Cp) is temperature-dependent, decreasing from -1.4 kJ K-1 mol-1 at 4 C to -2.9 kJ K-1 mol-1 at 30C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and theprotein-DNA complex are not changing in parallel when the temperature increases and that thermalmotions of the protein and the DNA are restricted in the complex. After correction for this effect, Cp is-1.8 kJ K-1 mol-1 and temperature-independent. However, this value is still higher than Cp of -1.2 kJK-1 mol-1 estimated by semiempirical methods from dehydration of surface area buried at the complexinterface. We propose that the discrepancy between the measured and the structure-based prediction ofbinding energetics is caused by incomplete dehydration of polar groups in the complex. In support, weidentify cavities at the interface that are large enough to accommodate ~10 water molecules. Our resultshighlight the difficulties of structure-based prediction of Cp (and other thermodynamic parameters) andemphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complexbut also of the folding thermodynamics of the protein and the DNA alone, which are described in theaccompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].